Due to their simplicity, speed, low cost and specificity, immunoassays have become useful tools for the analysis of a variety of biological substances and small compounds such as environmental pollutants. The vast majority of immunoassays for small analytes such as pesticides, industrial organic pollutants, microbial toxins, abused drugs, hormones, and pharmaceuticals etc., have a competitive format, i.e., once the anti-hapten antibodies are produced, the same hapten or a structurally related molecule is conjugated to a tracer enzyme for the competition with analyte for the binding sites of immobilized antibody or coating protein to capture free antibody in the competition with analyte. Competitive-format assays are inferior to noncompetitive formats for which immobilized antibody on the solid support captures target molecule and another antibody conjugated with a signaling molecule detects the captured molecule in terms of sensitivity, precision, kinetics and working range, and are more difficult to adapt to rapid “on site” or “clinic” assays, such as dipsticks or immunochromatography.
Traditional methods for developing immunoassays start with hapten synthesis and production of anti-analyte antibodies by immunization of animals with a hapten-protein conjugate. The hapten is typically an analyte related compound modified to be covalently conjugated to the carrier protein. Once an analyte-specific, high titer anti-hapten serum is obtained, a competitive immunoassay (e.g., an ELISA) can be developed by using the same hapten coupled to a unrelated carrier protein. When the same hapten is used for immunization and coating, the assay is designated as homologous ELISA. However, very frequently, homologous assays are less sensitive than heterologous assays (the hapten used for immunization and coating are different). The design of heterologous assays requires extensive chemical synthesis work in order to develop a proper panel of candidate haptens, which must afterwards be tested, to examine whether the desired sensitivity can be reached. For the detection of small molecules, a sandwich type noncompetitive ELISA format is not applicable because once antibody binds to the target molecule, there is no site available for the direct binding of secondary reporter antibody. Nevertheless, there have been efforts into the development of noncompetitive ELISA for small molecules and limited successes have been reported using anti-immunocomplex antibodies or recombinant antibody techniques. However, these methods require considerable time consuming and laborious procedures such as production of primary antibody against target molecule, reimmunization of analyte-antibody complex to obtain anti immunocomplex antibody, and screening of a panel of antibodies or generation of recombinant antibody library and successive screening to select the one with affinity for the analyte-antibody complex.
The compositions, methods and devices described herein provide improvements over current techniques for detecting small analytes, and allow the development of highly sensitive and specific assays for detecting small analytes in an easy to read noncompetitive format.